The objectives of this research are to determine the p21 GTP-binding site and the role of GTP binding in ras oncogene mediated-transformation. The ras oncogenes code for an approximately 21 kilodalton protein, p21, which, unlike other oncogene products, has a high specificity for the binding of guanine nucleotides. GTP-binding proteins are known to play major roles in transmembrane signaling, peptide synthesis, and cytoskeletal polymerization. These proteins manifest important similarities even though their predominant functions are markedly different. Studies whose experimental design have centered on potential analogous characteristics between these GTP-binding proteins have already proven to be extremely productive. Therefore, we will attempt to improve characterization of the ras family of oncogenes by exploring possible analogies with the better characterized GTP-binding proteins. It is anticipated that extension of this concept to p21 will likewise prove fruitful and result in more detailed information about the normal function of p21, the role p21 plays in oncogenesis, and the mechanisms(s) by which the normal cellular homologue of the ras oncogenes might become tumorigenic. The following specific areas will be investigated: (1)\utilizing computer DNA and protein databases, sequence homology between the ras oncogene system and (a)\other known oncogenes or (b)\other known GTP-binding proteins will be evaluated; (2)\site-directed mutagenesis of the cloned viral ras oncogenes will be done, and the ability of the mutant gene product to bind GTP and to induce transformation in NIH 3T3 cells will be evaluated; (3)\as p21 proteins are membrane bound, similarities between the viral p21 proteins and the membrane-bound GTP-binding protein from the light-activated phosphodiesterase (G-PDE) will be evaluated. We will determine the ability of p21-specific antibodies to immunoprecipitate G-PDE; the ability of p21 to replace the endogenous GTP-binding protein functionally; and, utilizing a GTP-photoaffinity analogue and two-dimensional electrophoresis, will evaluate similarities between the peptide maps. Promising protocols will then be analyzed for the GTP-binding protein from the hormone-activated adenylate cyclase system. And, (4)\if experiments at the protein level are fruitful, we will attempt to clone the gene for G-PDE by using a monoclonal antibody to enrich for mRNA and subsequently generate and clone the cDNA. (X)